This disclosure relates generally to nucleic acid analysis, and more specifically to nucleic acid synthesis using nanopores.
Currently available commercial platforms for sequencing DNA are relatively costly. These platforms use a ‘sequencing by synthesis’ approach, so called because DNA polymers are synthesized while detecting the addition of each monomer (i.e. nucleotide) to the growing polymer structure. Because a template DNA strand strictly directs synthesis of a new DNA polymer, one can infer the sequence of the template DNA from the series of nucleotide monomers that were added to the growing strand during the synthesis. The ability to detect monomer additions is facilitated by specially engineered variants of the biochemical components that normally carry out DNA synthesis in biological systems. These engineered components are expensive to make and are consumed in relatively large amounts during sequencing by synthesis. Furthermore, monitoring the reaction uses relatively expensive hardware such as lasers, detection optics and complex fluid delivery systems. The most successful commercial platforms to date also require expensive reagents and hardware to amplify the DNA templates before sequencing by synthesis can even begin.
Other sequencing methods have been considered in order to reduce cost, increase throughput, and/or simplify the process. One of these approaches is based on threading a single strand of DNA through a nanopore and identifying its sequence from the variation in the ionic current flowing through the pore as the strand is threaded. An alternative to this ‘nanopore-strand’ sequencing approach is ‘nanopore-exonuclease’ sequencing, which involves exonuclease catalyzed removal of nucleotide monophosphates, one at a time, from a DNA strand and sequentially passing the released nucleotide monophosphates through a nanopore. However, the resulting variations in the ionic current flowing through the nanopores are quite small and it is difficult to distinguish one nucleotide from another. Attempts have been made to modify the DNA before digestion or to modify the nucleotide monophosphates once they have been released. However despite these efforts, nanopore-exonuclease sequencing has not yet been demonstrated at a commercially viable level to date.
Thus, there exists a need for more cost effective, rapid and convenient platforms that provide an alternative to those currently available for sequencing nucleic acids. The present disclosure addresses this need and provides other advantages as well.